Abstract
Nanotechnology plays a promising role in biomedical applications,
particularly tissue engineering. Recently, the application of magnetic
scaffolds and pulsed electromagnetic field (PEMF) exposure has been
considered in bone tissue regeneration. In this study,
3rd generation dendrimer-modified superparamagnetic
iron oxide nanoparticles (G3-SPIONs) are synthesized comprehensively
characterized. Magnetic polycaprolactone (PCL) nanofibers are prepared
by incorporating G3-SPIONs within the electrospinning process ,and
physicochemical characteristics ,as well as cytocompatibility and cell
attachment are assessed. Eventually, the osteogenic differentiation
ability of adipocyte-derived mesenchymal stem cells (ADMSCs) cultured on
the magnetic scaffold with and without PEMF exposure was investigated by
measurement of alkaline phosphatase (ALP) activity and calcium content.
The expression of specific bone markers was studied using the Real-time
PCR method. According to the results, G3-SPIONs with mean size and zeta
potential of 17.95 ± 3.57 nm and 22.7 mV, respectively, show a high
saturation magnetization (57.75 emu/g). Adding G3-SPIONs to PCL
significantly decrease nanofibers size to 495±144 nm and improves cell
attachment and growth. The ADMSCs cultured on the G3-SPION-PCL scaffold
in the presence of osteogenic media (OM) and exposure to PEMF expressed
the highest Osteocalcin and Runx2 and showed higher calcium content as
well as ALP activity. It can be concluded that the synthesized G3-SPION
incorporated PCL nanofibers serve as a promising magnetic scaffold for
bone regeneration. Also, utilizing the magnetic scaffold in the presence
of OM and PEMF provides a synergistic effect toward osteogenic
differentiation of ADMSCs.
Key Words: Superparamagnetic iron oxide nanoparticles,
Dendrimer, Polycaprolactone, Pulsed electromagnetic field, Bone tissue
engineering
Introduction
According to the American academy of orthopedic surgeons, there are 6.3
million fractures every year in the United States. Although surgery
serves as a common treatment for bone fracture, in some cases (around
10%), infection and insufficient vascularization following the may
result in incomplete healing (Ng, Spiller,
Bernhard, & Vunjak-Novakovic, 2017;
Stevens, Yang, Mohandas, Stucker, &
Nguyen, 2008). The factors like aging populations and the high
incidence of osteo-degenerative diseases significantly lead to growth in
the artificial regeneration of bone tissue as a field of research
(Joshi & Grinstaff, 2008). Autologous
bone grafting is usually known as the gold standard for the treatment of
bone defects. However, there are several disadvantages, including
painful procedure, high cost, limited tissue supply, and extended
hospitalization (Kohli et al., 2018;
Polo-Corrales, Latorre-Esteves, &
Ramirez-Vick, 2014). On the other hand, despite the abundance of
resources for allograft based bone grafting, it has limited application
due to the poor adaptation and the risk of disease transmission
(Robbins, Lauryssen, & Songer, 2017;
Stevens et al., 2008). In order to
overcome such limitations, bone tissue engineering is becoming a
promising treatment in which the combination of living cells, engineered
scaffolds, and biochemical/biophysical factors can enhance tissue
regeneration. Actullay, bone issue engineering ,in association with
technologies in fracture stabilization, seems as innovative solutions
(Campana et al., 2014).
Recently, it has been documented that in addition to physical
stimulations including tensile and compressive stresses, fluid shear
stresses, and heat showing a remarkable role in osteogenic
differentiation, magnetic stimulations can also considerably improve
bone regeneration (Xia et al., 2018). In
this regards, magnetic nanoparticles (MNPs) incorporation into the bone
tissue engineering scaffolds, with or without a magnetic field exposure,
have enormous potential for bone tissue engineering applications
(Bock et al., 2010;
He et al., 2017). Previous studies have
demonstrated that ion exchanging channels on the cell membrane and the
corresponded biochemical pathway can be influenced by magnetic fields
(Zhao et al., 2011). Magnetic scaffolds
in which MNPs are incorporated have significant effects on adhesion,
proliferation, and differentiation potency of stem cells. The force
generated by magnetic nanoparticles under a magnetic field can
significantly influence the micro-environment around the cells, leading
to a series of changes in cell behavior via magneto-mechanical
stimulations (Jiang et al., 2016;
Xia et al., 2019;
Xia et al., 2018). Among MNPs, iron oxide
nanoparticles have attracted much interest in biomedical applications
such as magnetic-activated cell sorting (MACS), hyperthermia, drug
delivery, and contrast-enhanced MRI, because they belong to a group of
non-toxic materials that exhibit good biocompatibility due to the
presence of iron ions (Kandpal, Sah,
Loshali, Joshi, & Prasad, 2014) (Chauhan
et al., 2013; Ito, Shinkai, Honda, &
Kobayashi, 2005). (Ayyappan et al., 2010;
Koehler et al., 2009;
Marinin, 2012). As a challenging issue,
in biological media, MNPs usually tend to be agglomerated because of the
high surface-area-to-volume ratio as well as magnetic attraction. To
avoid this problem and to stabilize nanoparticles, the MNPs surface
coating is preferred (Mascolo, Pei, &
Ring, 2013). Ideally, these coatings should be non-immunogenic and
hydrophilic enough to prevent the opsonization by plasma proteins
resulting decrease in reticuloendothelial system clearance and an
increase in the shelf-life within the body. The materials used for
coating are commonly polymeric ones with improved physical-chemical
properties and appropriate biocompatibility
(Chauhan et al., 2013;
Favela-Camacho, Samaniego-Benítez,
Godínez-García, Avilés-Arellano, & Pérez-Robles, 2019). Dendrimers are
highlighted by their unique properties, such as mono-dispersity,
well-defined structure, having a large number of surface functional
groups, and antimicrobial activities. Such characteristics make them an
appropriate tool for biomedical applications, particularly tissue
engineering (Abdel-Sayed et al., 2016;
Gorain et al., 2017;
Kesharwani, Gajbhiye, K Tekade, & K Jain,
2011; Kesharwani, Tekade, Gajbhiye, Jain,
& Jain, 2011; Kesharwani, Tekade, &
Jain, 2015). Incorporation of dendrimers into the scaffolds structure,
particularly in the surface, may improve cell-substrate interactions.
Besides, the dendrimers due to the porous structure could improve the
interconnection in the scaffolds structure and consequently help to
cell-cell interactions (Gorain et al.,
2017; Joshi & Grinstaff, 2008). Poly
(amidoamine) dendrimers, PAMAM, have received widespread attention
because of the fundamental nature and their polar properties
(Bosman, Janssen, & Meijer, 1999). PAMAM
are hydrophilic, biocompatible, monodisperse, and cascade-branched
macromolecules with highly flexible surface chemistry. In order to
reduce MNPs agglomeration and increase their cationic surface charge,
coating with PAMAM can be considered as an ideal option because PAMAM
has plenty of peripheral functional groups and high hydrophilicity.
PAMAM dendrimers can introduce a dense outer amine shell on the MNPs
through a cascade-type generation (Boas,
Christensen, & Heegaard, 2006;
Khodadust, Unsoy, Yalcın, Gunduz, &
Gunduz, 2013; Klajnert & Bryszewska,
2001). Polycaprolactone (PCL) is considered as an appropriate candidate
for bone tissue engineering regarding its long-term degradation. the FDA
approves PCL for some clinical applications. It is biodegradable
polyester and appropriate for long-term bone implantation. Studies have
shown that PCL act as a supportive role to keep living osteoblasts and
fibroblasts cells. PCL maintains its primary structure in the biological
fluids and tends to blend with other polymers. [31] Considering the
inherent properties of MNPs, positive features of dendrimers as a right
candidate for surface coating, this project aimed to synthesize a
PCL-based magnetic scaffold by incorporation of dendrimers-modified
MNPs. Iron oxide nanoparticles (SPION) were functionalized by
3rd generation of PAMAM dendrimer and comprehensively
characterized. Dendrimerized SPIONs were incorporated into the PCL
nanofibrous scaffold by blend electrospining. Osteogentic
differentiation of MSCs cultured on the magnetic scaffolds was studied
in the presence of pulsed electromagnetic field produced by bioreactor.
Materials and methods
2.1. Chemicals
Ferrous chloride hexahydrate
(FeCl3.6H2O), ferric sulfate
heptahydrate (FeSO4.7H2O),
(3-aminopropyl) triethoxysilane (APTES), methanol
(CH3OH), ethanol
(C2H5OH), Chloroform (CHCl₃), dimethyl
sulfoxide (DMSO) and beta-glycerol phosphate were purchased from Merck
(Darmstadt, Germany). Methyl acrylate (MA), ethylenediamine (EDA),
ammonium hydroxide (NH4OH), polycaprolactone (PCL,
MW= 70000-90000 g/mol), dimethylformamide (DMF),
3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT),
dexamethasone, ascorbic acid-2-phosphate, glutaraldehyde,
paraformaldehyde, and diarylpyrimidine (DAPI) were purchased from
Sigma-Aldrich (St. Louis, MO) and used without further purification.
Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),
penicillin/streptomycin were purchased from Gibco (USA). Human adipose
mesenchymal stem cells (ADMSCs ) obtained from Stem Cells Technology
Research Centre cell bank (Tehran, Iran).
2.2. Synthesis of SPIONs
The co-precipitation method was used to synthesize SPIONs following the
reported standard protocol. (Esmaeili,
Khalili, et al., 2019; Tajabadi,
Khosroshahi, & Bonakdar, 2013) A solution mixture of ferrous chloride
hexahydrate (FeCl3.6H2O), 0.2 M, and
ferric sulfate heptahydrate
(FeSO4.7H2O), 0.1 M, was prepared in
distilled water and used as a source of iron ions. The aqueous ammonia
solution (5.4 M) was added dropwise to the solution of iron salts under
nitrogen protection and stirred at 50 °C for 30 min. The obtained
precipitation was washed five times with distilled water using magnetic
separation to remove any excess unreacted materials and air-dried at
room temperature.